Circadian regulation of immune system function is poorly understood but has significant impact on human health. Many measures of immune system function, such as baseline levels of the cytokines TNF and IFN- gamma, vary with time of day. Loss of circadian regulation is also associated with poor immune system function and susceptibility to infection. We have established a system to study circadian-regulated immune function in Drosophila. We found that flies infected at different times of day with S. pneumoniae die with different survival rates and that this is due to the effects of circadian regulatory protens. We further identified a specific immune response that is circadian-regulated: phagocytosis of bacteria. Though the cell biology of phagocytosis is well studied in unicellular organisms and in cultured cells, the molecular regulation of phagocytosis in multicellular organisms is less clear. Similarly, though much is known about circadian regulation, the major focus has been on molecular mechanisms and circadian regulatory neurons (pacemaker neurons)-not on effectors tissues. We need to understand the circadian regulation of relevant effectors tissues to translate these discoveries into medical practice. In preliminary data, we found that inhibition of the circadian regulator Clock in either pacemaker neurons or phagocytic immune cells increases resistance to infection by S. pneumoniae. We hypothesize that pacemaker neurons regulate the circadian rhythms of phagocytic immune cells, dictating the oscillation of Clock activity in those cells as well as phagocytic activity. In this proposal, we will test this hypothesis in three ways: we will identify cellular mechanisms underlying circadian-regulated phagocytosis (Aim 1); we will also examine how cell-autonomous circadian regulators control phagocyte function (Aim 2) and how pacemaker neurons non cell-autonomously regulate phagocyte function (Aim 3). Thus the proposed experiments will analyze circadian regulation of immune system function on molecular, cellular, and systemic levels. Because of the high evolutionary conservation of both innate immunity and circadian biology, defining the molecular mechanisms in Drosophila will provide insight into ways to improve human innate immune system function.